Anti ha antibody 12ca5

Anti—c-Maf (M-153), anti-Hsp90 (sc-7947) and anti-Oct1 (sc-232) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated 4G10 (anti-phosphotyrosine), HRP-conjugated goat anti-mouse and goat anti-rabbit L chain antibodies were purchased from Millipore (Billerica, MA). Anti-p-Tyr (P-Tyr-1000) antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-HA (12CA5) antibody and Protein A and G agarose beads were purchased from Roche (Mannheim, Germany). Anti-FLAG (M2) antibody was purchased from Sigma-Aldrich (St. Louis, MO). Anti-EGFP (ab290) antibody was purchased from Abcam (Cambridge, MA). Anti-mouse Ptpn22 (anti-PEP) antibody was obtained from Genentech (San Francisco, CA). Anti-Tec antibody was purchased from Upstate biotechnology. Anti-α-Tubulin 4a antibody was purchased from GeneTex. Antibodies to CD3-ε (17A2), CD28 (37.51), IL-4 (11B11), and IFNγ (XMG1.2) were purchased from BioLegend (San Diego, CA). Recombinant human IL-2 and TGF-β and recombinant mouse IL-4 and IL-6 were purchased from PeproTech (Rocky Hill, NJ). Recombinant mouse IL-23 was purchased from eBioscience (San Diego, CA).

For Chk1 and Cds1 phosphorylation analysis, exponentially growing cells in YES media were treated with 200 μM K₂Cr₂O₇, NaAsO₂ or CdSO₄ for 2 or 4 hours at 30°C. Cells were also treated with 20 mM HU or 30 μM CPT for positive controls. Whole-cell extracts from 50 ml cultures were resuspended in standard lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 2.5 mM EDTA, 0.002% NP-40, 50mM NaF, 1X Roche cOmplete protease inhibitor cocktail) and disrupted with glass beads in an MP Biomedicals FastPrep-24 instrument using 4 bursts of 20 seconds at a speed setting of 5.0. Samples containing 120–150 mg of protein were resolved on 10% SDS-PAGE with acrylamide/bisacrylamide ratio of 99:1. Proteins were transferred to nitrocellulose membranes, blocked with 5% milk in TBS buffer with 0.05% Tween and probed with an anti-HA (12CA5) antibody (Roche). Anti-α-Tubulin from Sigma (T5168) was used as loading control.

HA-K-Ras(V12), HA-H-Ras(V12), and HA-N-Ras(V12) were expressed in baculovirus-infected Sf9 insect cells and purified from the particulate (P100) membrane-containing fraction with anti-HA (12CA5) antibody (Roche, Mannheim, Germany) covalently linked to Protein A Sepharose® Cl-4B (GE Healthcare, Braunschweig, Germany). Purified HA-tagged Ras proteins (500 ng) were immunoprecipitated with μMACS HA Isolation Kit as described in Meinohl et al., 2019 [31 ]. Precleared PANC-1 cell lysate (10 mg) was added to immobilized HA-Ras proteins and incubated for 2 h under constant rotation. Protein complexes were purified on μColumns (Miltenyi Biotec, Germany) as described in Meinohl et al., 2019 [31 ]. The eluted proteins were separated on 10% SDS-polyacrylamide gels and either silver-stained or subjected to Western blotting using anti PI3-K p85α antibody and ECL detection.

Protein extracts (6–10 mg) were incubated with 10 μl of Anti-HA 12CA5 antibody (Roche, #11583816001) for 1 h at 4 °C, then 25 μl of Protein a Agarose was added (Roche, #11134515001). Samples were incubated overnight with orbital rotation at 4 °C. Beads were washed six times with co-IP Washing Buffer (50 mM Tris–HCl, pH 7.5; 500 mM sodium chloride; 0.1% Nonidet P-40). Bound proteins were solubilized by the addition of SDS-sample buffer and heating at 95 °C for 8 min. Alternatively, HA-tagged proteins were immunoprecipitated using anti-HA Magnetic beads (Pierce, #88836). After 2 h incubation at 4 °C, beads were washed six times using a magnetic rack with co-IP Washing Buffer. Bound proteins were solubilized with 25 μl of HA Synthetic Peptide (Sigma, #11666975001) at 37 °C for 30 min. For pulling down GFP-tagged proteins, 6–10 mg total protein were incubated with 10 μl with Anti-GFP (Roche, 11814460001) for 1 h at 4 °C and, subsequently, 25 μl of Dynabeads Goat Anti-Mouse IgG (Invitrogen, #11,033) was added. Samples were incubated overnight with orbital rotation at 4 °C. Magnetic beads were washed six times using a magnetic rack with co-IP Washing Buffer. Bound proteins were solubilized by the addition of SDS-sample buffer heated at 95 °C for 8 min.

For Chk1 shift, whole cells extracts were prepared from exponentially growing cells in standard NP-40 lysis buffer. Protein amounting to ~100 mg was resolved by SDS-PAGE using 10% gels with acrylamide:bis-acrylamide ratio of 99:1. Proteins were transferred to nitrocellulose membranes, blocked with 5% milk in TBST (137 mM Sodium Chloride, 20 mM Tris, pH 7.6, 0.05% Tween-20) and probed with anti-HA (12CA5) antibody (Roche).